1. Field of the Invention
This invention generally relates to Bowman-Birk Inhibitor (BBI) and more particularly to a Bowman-Birk Inhibitor Concentrate (BBIC) extracted from soybeans, a method of producing a Bowman-Birk Inhibitor Concentrate (BBIC) and products containing the Bowman-Birk Inhibitor Concentrate (BBIC).
2. Description of the Related Art
Bowman-Birk Inhibitor (BBI) is an enzyme-inhibitor described by Bowman (Proc. Soc. Exptl. Med., 63:547 (1946)) and Birk et al. (Bull. Res. Council Israel, Sec. A 11:59 (1962) and Biochem. Biophys. Acta, 67:326 (1963)), and is found in crude soybean extract. Bowman-Birk Inhibitor (BBI) has been shown to exhibit inhibitory activity against the malignant transformation of cells under certain conditions and its administration has been shown to affect various forms of cancer. It has been demonstrated that the Bowman-Birk Inhibitor (BBI) exhibits certain physiological activity that prevents, or at least greatly reduces, radiologically or chemically induced malignant transformation of cells in culture and in laboratory animals. Yavelow et al. (Proc. Natl. Acad. Sci., USA 82:5395–5399 (1985)) reported that a crude soybean extract, if defatted with acetone, effectively blocked cell transformation in vitro. An active component of this crude extract is the BBI. These observations, along with epidemiological data, suggest the use of BBI as a putative dietary anticarcinogen, particularly with respect to colon cancer.
Weed et al. (Carcinogenesis, 6:1239–1241 (1985)) disclose that an extract of soybeans containing the Bowman-Birk protease inhibitor, when added to the diet of dimethylhydrazine (DMH)—treated mice, resulted in significant suppression of odenomatous tumors of the colonic mucosa. DMH—induced colon cancer in mice is generally regarded as an excellent animal model for the human disease, because the DMH carcinogen treatment induces adenocarcinomas of the mouse colon and rectum, which are similar to the tumors arising in the human colon. These results suggest that a dietary additive of the sort studied might confer some protection against the development of human colon cancer without undesirable side effects. The BBI extract and methods for its preparation were as described by Yavelow et al. Cancer Res., 43:2454–2459 (1983); Proc. Natl. Acad. Sci., USA 82:5395–5399 (1985) and Hwang et al. Biochem. Biophys. Acta, 495:369–382 (1977).
Massadi et al. (JNCI, 76:447–452 (1986)) demonstrated that a soybean extract containing the protease inhibitor BBI suppresses 7,12-dimethyl-benz[a]anthracene (DMBA)—induced carcinogenesis in the hamster cheek pouch. This oral cancer model, with the use of the hamster cheek pouch carcinogenesis system, has the same histopathology, growth pattern, and precancerous lesions as the most common form of human oral cancer, squamous cell carcinoma. This study demonstrates that hamster cheek pouch carcinogenesis can be inhibited by BBI and suggests that human oral carcinogenesis might respond to BBI in a comparable manner. The BBI preparation used in this study was a crude extract of the inhibitor prepared as described by Yavelow et al. (Proc. Natl. Acad. Sci., USA 82:5395–5399 (1985)).
Baturay et al. (Cell Biology and Toxicology, 2:21–32 (1986)) disclose that a BBI preparation, derived from a crude soybean extract defatted with acetone, suppresses radiation and chemically induced transformation in vitro, with or without enhancement by the co-carcinogen, pyrene. Yavelow et al., 1985, supra show that either pure BBI or the BBI extract prepared in accordance with their methods suppresses radiation-induced transformation in C3H10T½ cells. Kennedy et al, Proc. Nat'l. Acad. Sci. USA 1984, 81, 1827–39, report that either pure BBI or the BBI extract prepared in accordance with their method reduce the levels of chromosome abnormalities in cells of patients with Bloom's syndrome (a genetic disease in which the high levels of chromosome abnormalities are thought to predispose the patients to a higher than normal cancer incidence). Still, other studies suggest that soybean-derived protease inhibitors can have suppressive effects on skin, breast, and liver carcinogenesis in vivo.
Kennedy et al. in Anticarcinogenesis and Radiation Protection, edited by Cerutti et al., Plenum Pub. Co., pp. 285–295 (1987), disclose that carcinogenesis in various systems is suppressed using a crude BBI extract prepared by defatting soybeans with acetone. Their results suggested that very low concentrations of BBI-type protease inhibitor preparation would be effective as chemopreventative agents for colon cancer. There was no evidence to suggest that the use of protease inhibitors as chemopreventative agents would be complicated by possible toxicity problems.
St. Clair et al. (Cancer Res., 50:580–586 (1990)) report that the addition of 0.5% or 0.1% semi-purified BBI or 0.1% or 0.01% purified BBI to the diet of DMH-treated mice resulted in a statistically significant suppression of angiosarcomas and nodular hyperplasia of the liver and colon carcinogenesis. The results of this study also indicate that BBI, included as 0.5% of the diet or less, had no adverse effect upon the health of the mice, but had capacity to suppress liver and colon carcinogenesis.
Perlmann et al. (Methods in Enzymology, 19:860–861 (1970)) have described an elaborate method for obtaining the BBI from a defatted soybean extract.
U.S. Pat. No. 4,793,996 to Kennedy et al. discloses a process comprising treating soybeans with acetone, followed by ethanol extraction and acetone precipitation for obtaining BBI. The soybeans may be defatted prior to acetone treatment. In addition, BBI may be further purified by conventional techniques. Kennedy et al. discovered that in the conventional process for preparing BBI from soybeans, a factor remained which adversely affected the ability of BBI to inhibit the malignant transformation of cells. If the factor was removed, the resulting BBI product was capable of inhibiting the malignant transformation of cells. It was found to be possible to remove this factor by treating the soybeans with acetone prior to the ethanol extraction step taught by Perlmann et al. (supra)
Kennedy et al. teach that it is unnecessary to carry out a procedure requiring complete purification of the extract to the point where the product contains only a single protein, but instead determined that it was sufficient to stop the purification procedure at a point where a crude inhibitor extract is obtained. This crude extract (i.e., concentrate) is itself edible and can be used as an inhibitor of malignant transformation of cells by oral ingestion. Kennedy et al. disclose a process for preparing a crude soybean extract containing an inhibitor of malignant cell transformation which comprises defatting soybeans by bringing them into contact with at least an equal weight of acetone, and extracting the inhibitor from the defatted soybeans with alcohol, thus producing a crude inhibitor extract having greatly increased effectiveness.
U.S. Pat. No. 5,217,717 to Kennedy et al. discloses a method for producing BBI concentrate from soybean solubles without aqueous alcohol extraction by centrifugation and ultrafiltration, and further purification by acetone precipitation. This patent also teaches that the less solvent used, the more economical and safer the process. Waste-solvent streams containing a mixture of alcohol-water-acetone require very complex and expensive distillation equipment for separation of the solvents.
The presence of BBI is commonly measured by the level of chymotrypsin inhibition (CI). The present invention provides a new method for making an acetone-extracted Bowman-Birk Inhibitor concentrate having at least 50% soy protein by weight of dry matter and a chymotrypsin inhibitor (CI) level of at least 200 milligrams/gram.